This invention relates t o methods and formulations for mediating virus attachment and infection, and more particularly relates to methods and formulations for mediating adeno-associated virus attachment and infection.
Adeno-associated virus (AAV) is a human parvovirus that infects a broad range of cell types including human, non-human primate, canine, murine, and avian. A member of the Parvoviridae family, AAV is a small non-enveloped single-stranded DNA virus of 20-25 nm which has an unique requirement for a helper virus (e.g., adenovirus or herpes simplex virus) to complete its lytic cycle (R. W. Atchison et al., (1965) Science 149:754; M. D. Hoggan et al, ((1966) Proc. Natl. Acad. Sci. USA 55:1457; J. L. Melnick et al., (1965) J. Bacteriol. 90:271). In the absence of helper virus, AAV still infects the target cell, but integrates into the host genome and establishes latency. Unique among eukaryotic DNA viruses, the AAV genome can integrate site specifically into human chromosome 19 (R. M. Kotin et al., (1990) Proc. Natl. Acad. Sci. USA 87:2211; R. J. Samulski et al., (1991) EMBO J. 10:3941; R. J. Samulski, (1993) Curr. Opin. Genet. Dev. 3:74; C. Giraud et al., (1994) Proc. Natl. Acad. Sci. USA 91:10039; C. Giraud et al., (1995) J. ViroL 69:6917). This property has drawn considerable attention to the potential use of AAV as a gene therapy vector, although little is known about the initial events of AAV infection (R. J. Samulski, (1995) Adeno-associated virus-based vectors for human gene therapy, p. 232-271. In K. M. Hui (ed.), Gene therapy: from laboratory to the clinic. World Scientific Publishing Co., Singapore, Singapore; C. McKeon et al., (1996) Hum. Gene Ther. 7:1615; D. M. McCarty et al., (1997) Adeno-associated viral vectors, p. 62-78. In M. Strauss and J. Barranger (ed.), Concepts in gene therapy. Walter de Gruyter, Bellin. N.Y.; R. J. Samulski, (1997) Development of adeno-associated virus as a vector for in vivo gene therapy, p. 197-203. In L. M. Houdebine (ed.), Transgenic animals: generation and use. Harwood Academic Publishers, Chur, Switzerland). In particular, the recombinant AAV or rAAV vector system is well characterized and is the subject of increasing development as a vector for gene delivery (see, C. McKeon et aL (1996) Hum. Gen. Ther. 7:1615). In general, AAV vectors are generated by deleting rep and cap genes and replacing them with genes intended for delivery into the cell. Additionally, producer cells that contain rep and cap may be used to package the gene therapy vectors into the AAV capsid particle (B. J. Carter, (1996) Nature Biotechnology 14:1725).
Despite this growing interest in AAV, the events that govern the initial AAV infection remain poorly understood. The primary event of any viral infection is attachment of virus to the host cell. A wide variety of cell surface molecules are now known to serve as viral attachment receptors. However, the mechanism by which AAV attaches to its host cell has heretofore not been delineated. AAV has a very broad host range and infects a wide variety of cell types, suggesting that the virus uses a ubiquitous receptor to mediate infection. Identification of the initial virus-host cell interactions necessary for efficient AAV infection is not only important for the general understanding of parvovirus infection, but also for the effective use of AAV as a gene therapy vector.
Although the initial events in the life cycle of AAV are not well understood, previous studies suggest that AAV infects cells through interaction with a specific host cellular receptor (H. Mizukami et al., (1996) Virology 217:124; S. Ponnazhagan et al, (1996) J. Gen. Virol. 77:1111). AAV appears to exhibit saturation binding to HeLa cells. In addition, cellular attachment of AAV is sensitive to trypsin treatment, suggesting a protein component is responsible for binding. Id.
The lack of knowledge concerning the receptor of AAV has introduced significant obstacles to the development of reliable techniques for both isolating and using AAV as a means for gene therapy. For example, purification of AAV is generally conducted using techniques that ultimately involve the use of a CsCl gradient. There are certain disadvantages in using these techniques, primarily because CsCl is toxic and thus requires special handling. It would be highly desirable to develop a milder and less dangerous means of isolating AAV viral particles.
An additional obstacle to the use of AAV as a reliable gene therapy vector has been the difficulty in infecting certain types of cells with the vector. Experiments in cultured cells have shown that AAV vectors are efficient for delivery of genes to both dividing and non-dividing cells. However, these experiments have also shown that the efficiency and both expression and metabolic activation may vary with the cell type and the physiological state of the cell (C. McKeon et al., (1996) Hum. Gen. Ther. 7:1615). In particular, progenitor or stem cells (e.g., bone marrow CD34+ cells) have been found to be difficult to infect with the AAV vector. Additionally, in some cell types, persistence and expression of a heterologous gene carried by the vector are not well maintained. Finally, even when it is known that certain cell types are generally permissive to infection by AAV, is appears that there is diversity among individual cell donors as to whether or not any particular donor""s cells will permit infection by the AAV vector. It would be highly desirable to have means for the effective infection of stem cells and rare cell types, as well as the means for introducing the AAV vector into cells that may not naturally express the AAV receptor, or may not naturally produce the molecular substituents necessary for the attachment and internalization of the virus.
Accordingly, there is a need in the art for improved methods and reagents for purifying AAV and rAAV vectors. In addition, there is a need in the art for methods of modifying the wild-type tropism of AAV vectors for use in gene therapy and for screening cells for permissiveness to transduction by AAV vectors.
The methods, AAV vectors, and formulations of the present invention are based on the surprising discovery that has identified cell surface heparin and heparan sulfate (HS) proteoglycan as the primary cellular receptors for AAV. It has also been discovered that AAV interacts specifically with cell surface heparin and heparan sulfate glycosaminoglycans (GAG), and not other glycosaminoglycans. Further, it has now been determined that the presence of HS GAG on the cell surface directly correlates with the efficiency by which AAV can infect cells.
Moreover, a role has been established for xcex1vxcex25 integrin in AAV infection. AAV virions physically interact with the xcex25 subunit of xcex1vxcex25 integrin. Using genetically defined cell lines that either lack or express xcex1vxcex25, it has been demonstrated that cell surface expression of this integrin promotes AAV infection. The present investigations suggest that xcex1vxcex25 integrin acts to facilitate the internalization of AAV bound to cell surface heparin and HS proteoglycans into the cell. This is the first report of the involvement of an integrin in a parvovirus infection.
These discoveries have led to the development of methods and formulations that mediate the infection of a broad range of cell types with AAV, including cells that are typically non-permissive for infection by AAV. Additionally, these discoveries have led to the development of methods of purifying AAV using receptor-like molecules that bind to AAV, and methods of screening cell samples for their permissiveness to infection with AAV. Furthermore, these discoveries have elucidated new strategies for modifying the natural tropism of AAV, in particular, for use in gene therapy.
Accordingly, a first aspect of the present invention is a method of facilitating attachment of AAV to a cell, and infection of a cell by AAV, by contacting the cell with a soluble artificial receptor or soluble receptor-like molecule that mediates attachment and infection of AAV into the cell. This aspect of the invention is based on the observation that low concentrations of soluble heparin, HS and high molecular weight dextran sulfate enhance AAV infection. Heparin, HS, and other polyanionic molecules are known to attach to the cell surface. Therefore, exogenous heparin, HS, GAGs and other polyanionic molecules (preferably, heparin and HS) can mediate AAV attachment to and infection of cells that do not typically express heparin or HS on the cell surface (or that express these molecules at low concentrations).
The discovery that heparin and HS proteoglycans are the receptor for AAV has also led to the development of a further aspect of the present invention, which is a method of purifying and/or concentrating AAV. According to one embodiment, a receptor-like molecule is immobilized to a matrix to form a solid support that binds the AAV. Samples suspected of containing AAV are then contacted with the immobilized receptor-like molecules. The bound AAV is eluted (e.g., with a high salt wash) and collected. This method may be practiced in numerous alternative embodiments, for example, by affinity chromatography, by batch purification methods (e.g., with magnetized beads), or by immobilizing the receptor-like molecule to a polymeric surface such as a plate or a tube. As a further alternative, the matrix can be a material such as fiberglass, cellulose acetate, nitrocellulose, or nylon. Such matrices can be advantageously employed to bind AAV, e.g., for titering or purification for analytical purposes.
The receptor of AAV having been determined relates to the a further aspect of the present invention, which is a method of facilitating or enhancing attachment of AAV to a cell, thus increasing the efficiency of AAV infection into a cell. In one particular embodiment of this method, the AAV capsid is mutated using techniques known to those skilled in the art, such that the mutant AAV exhibits enhanced attachment to cellular receptors and thus may increase infectivity of the AAV into the cell. More particularly, at least one of the AAV binding sites for heparin/HS is mutated, such that binding is enhanced. According to another embodiment, binding of AAV to a cell is facilitated or enhanced by upregulating the expression of receptors (e.g., heparin or HS) on the surface of the cell. Exemplary compounds that upregulate cell surface expression of heparin and HS are transforming growth factory, sodium butyrate, and fibroblast growth factor.
A further aspect of the present invention is a method of inhibiting or preventing binding of AAV to a cell. In one embodiment of this method, the AAV is mutated using techniques known to one skilled in the art, such that binding of AAV is prevented or inhibited. In particular, at least one of the AAV binding sites for heparin and/or HS is mutated (e.g., by deletion or by replacing basic amino acids with neutral amino acids) such that binding of AAV to cell surface receptors is prevented or inhibited. In another embodiment of this method, a cell that naturally expresses the AAV receptor is treated with an enzyme or reagent that removes or alters the natural AAV receptor, such that AAV binding to the cell is prevented or reduced or the AAV receptor can no longer mediate infection of the cell by AAV. In yet another embodiment of this method, AAV virus is treated with molecules (e.g., heparin, HS, high molecular weight dextran sulfate, antibodies) that have been determined to block the interaction between AAV and the AAV receptor at concentrations effective to inhibit or prevent binding of AAV to the cell, compared to that which would occur in the absence of such treatment.
A further aspect of the present invention is a method of screening a cell for permissiveness to AAV infection by detecting the presence or absence, or alternatively, the abundance, of the AAV receptor on the cell surface. In this method, a cell or sample of cells is contacted with, for example, an antibody to the AAV receptor. Binding of the receptor to the antibody is then detected and visualized by techniques that are readily available to one skilled in the art. This method finds particular use in screening potential donors for cells that may be used in gene therapy, in screening recipients for permissiveness to gene therapy using an AAV vector, and in screening cells for potential use as producer cells for the AAV vector.
A further aspect of the present invention are formulations containing AAV vectors. In one embodiment, the present invention provides formulations useful in the mediation of cell attachment to, or infection by, AAV. The formulation contains an AAV vector along with a soluble receptor-like molecule or artificial receptor of the present invention, preferably in a physiologically or pharmaceutically acceptable carrier. The AAV vector in such a formulation may optionally contain mutations in the binding site for the receptor that enhance binding to the receptor. A second embodiment is a formulation useful in preventing or inhibiting binding of the AAV vector to a cell comprising an AAV vector along with a molecule that blocks binding of the vector to the natural receptor. This formulation will aid in specific targeting of AAV vectors. The AAV vector in such a formulation may optionally contain mutations in the binding site for the receptor that inhibit binding to the receptor. Formulations of the present invention may optionally contain certain additives such as stabilizers or protease inhibitors known to one skilled in the art. Furthermore, AAV vectors provided in formulations of the present invention may optionally comprise heterologous genes that are to be delivered into a target cell for the purpose of expressing the heterologous gene in the cell, e.g., for gene therapy.
A further aspect of the present invention is a kit for mediating AAV attachment to, and infection of, a cell. Such a kit will comprise an AAV vector along with at least one compound that mediates AAV attachment to and infection of a cell, preferably packaged together in a container with written instructions for using the kit.
A further aspect of the present invention is a kit for screening cell samples for permissiveness to AAV infection. Such a kit will comprise a first reagent that binds to the AAV receptor, such as an antibody to the receptor, along with a second reagent for detecting binding between the AAV receptor and the first reagent. The reagent that specifically binds to the AAV receptor and the detecting reagent are preferably packaged in a single container along with written instructions for using the components of the kit to determine if a cell sample is permissive for AAV attachment and infection.
A further aspect of the present invention is a method of enhancing the delivery and transduction of a heterologous gene into a cell, wherein the heterologous gene is delivered into a cell by an AAV vector. In such a method, the heterologous gene is carried by an AAV vector produced using methods known to those skilled in the art. In the present invention, the AAV vector is contacted with the target cell, wherein the target cell is exposed to a soluble receptor-like molecule or artificial receptor of the present invention. In an alternative embodiment, the AAV vector carrying the heterologous gene is contacted with the cell simultaneously with the soluble receptor like molecule.
The discovery that xcex1vxcex25 integrin serves as a co-receptor to facilitate infection by AAV is related to a further aspect of the invention, which is a method of facilitating or enhancing infection of AAV into a cell by treating the cell with a compound that induces or enhances the expression of integrin (preferably, xcex1vxcex25 integrin) on the surface of the cell. Illustrative compounds for upregulating cell surface integrin include cytokines (including interleukins, e.g., IL-1b), hematopoietic growth factors, (e.g., granulocyte-macrophage colony stimulating growth factor and macrophage colony stimulating growth factor), and phytohemagglutinin. As a further aspect, also provided are methods of screening a cell or cell sample for permissiveness to infection by AAV by detecting the presence or absence (or alternatively, the abundance) of integrin (preferably, xcex1vxcex25 integrin) expression on the surface of the cell(s). Also provided, as a further aspect, is a kit for determining if a cell is permissive for infection by AAV, where the kit provides reagents for detecting the presence or absence (or alternatively, abundance) of integrin (preferably, xcex1vxcex25 integrin) on the cell surface.
These and other aspects of the present invention will be set forth in more detail in the description of the invention below.
FIGS. 1A and 1B illustrate the inhibition of AAV infection by various glycosaminoglycans. In the data shown in FIG. 1A, rAAV was incubated with the indicated concentrations of heparin (solid squares), chondroitin sulfate B (dermatan sulfate) (open circles), chondroitin sulfate A (open diamonds) or chondroitin sulfate C (open triangles) for 1 hour at 37xc2x0 C. prior to a 1-hour adsorption period of the virus/GAG mixture to HeLa cells for infection. xcex2-galactosidase activity was assayed 44 hours post-infection using a Galacto-Light Plus kit (Tropix Inc.), and measured in a luminometer. Each point denotes the average % decrease in Relative Light Units (RLU) per ug of protein relative to the maximum RLU/xcexcg protein obtained in experiments without GAG. In the data shown in FIG. 1B, HeLa cells were preincubated with increasing concentrations of heparin at 37xc2x0 C. for 1 hour. After thorough washing, cells were infected with rAAV as described above. Data points represent the average % maximum RLU/ug protein obtained without heparin preincubation.
FIG. 2 illustrates that soluble heparin inhibits binding of AAV to the cell surface. After pre-incubation of 3H-wtAAV-2 with increasing concentrations of the indicated GAGs or the GAG analogue dextran sulfate, labeled virus was adsorbed to HeLa cells for 90 minutes at 4xc2x0 C. Unbound virus was removed by three washes with ice cold binding buffer and radioactivity was quantitated as described in methods. Data is represented as the average % inhibition relative to the CPM bound in the absence of soluble GAG.
FIGS. 3A, and 3B illustrate the effect of enzymatic digestion of cell surface glycosaminoglycans on AAV binding and infection. In the data shown in FIG. 3A, HeLa cells were treated with the indicated concentrations of the following GAG lyases: heparitinase (solid squares), heparinase I (open diamonds), chondroitinase ABC (open circles), or chondroitinase AC (open triangles), as described in Example 13, below. After thorough washing, the ability of AAV to bind the cell surface was assessed as described in Example 12, below. Data points represent the average % reduction in AAV binding relative to AAV binding obtained without enzymatic treatment. In the data shown in FIG. 3B, HeLa cells were treated with heparitinase or heparinase I as described herein. After thorough washing, rAAV was incubated with cells for a 1 hour adsorption period at 37xc2x0 C. Cells were harvested 44 hours post-infection and assayed for xcex2-galactosidase activity. Results are shown as the average % reduction in AAV transduction relative to transduction observed in the absence of enzymatic treatment. Data points represent the mean and standard deviation of experiments performed in triplicate.
FIGS. 4A and 4B illustrate that heparan sulfate proteoglycan serves as a primary attachment receptor for AAV-2. Wild type CHO-K1 cells and CHO-K1 mutants defective in proteoglycan synthesis were assessed for their ability to bind AAV-2. The cell line pgsA-745 lacks heparan sulfate and chondroitin sulfate proteoglycans. The cell line pgsD-677 lacks heparan sulfate proteoglycan and produces three fold excess chondroitin sulfate proteoglycans. The cell line pgsB-618 produces 15% of normal proteoglycans; while the cell line pgsE-606 produces an undersulfated form of heparan sulfate proteoglycan and normal levels of chondroitin sulfate proteoglycans. As shown in FIG. 4A, fluourescently labeled AAV-2 was bound to wild type CHO cells (Panel I) and the pgsA-745 mutant that lacks proteoglycans (Panel II) as described in methods. Images were captured using confocal microscopy. FIG. 4B illustrates binding of 3H-AAV to parental and mutant CHO cells. Binding assays were performed at 4xc2x0 C. in Eppendorff tubes. 3xc3x97105 cells were incubated with 4xc3x971011 particles of 3H-AAV for 90 minutes in HBS binding buffer. After thorough washing, cells were pelleted, solubilized and radioactivity quantitated as described in methods. Non-specific binding was determined by parallel binding studies done in the presence of 100 fold excess unlabeled virus. Data represent the mean specific binding and standard deviation obtained from experiments performed in triplicate.
FIGS. 5A and 5B illustrate that heparan sulfate proteoglycan mediates AAV infection. FIG. 5A graphically illustrates AAV infection of wild type and mutant CHO cells deficient in proteoglycan synthesis. rAAV-LacZ virus was incubated with cells at an MOI of 10 for 1 hour at 37xc2x0 C. Cells were harvested 44 hours post- infection and assayed for xcex2-galactosidase activity. Data represent the mean and standard deviation of triplicate experiments. FIG. 5B illustrates UV treatment of wild type and mutant CHO cells and its effect on rAAV transduction. Cells were treated with 45 Joules/m2 UV in a UV stratalinker (Stratagene) prior to infection with rAAV-LacZ as described above. xcex2-galactosidase activity was measured as described for non-UV treated cells.
FIG. 6 illustrates that low concentrations of heparin enhance rAAV transduction of HeLa cells. HeLa cells were infected with rAAV-LacZ virus in the presence or absence of heparin, at the indicated concentrations, for one hour at 37xc2x0 C. 48 hours after infection, cells were harvested and assayed for xcex2-galactosidase activity, which is indicates as relative light units (RLU)/xcexcg protein. Results are reported as the mean and standard deviation of one experiment performed in triplicate.
FIG. 7 demonstrates that HSPG is an important determinant of AAV type 3 infection. AAV3-LacZ virus was incubated with CHO cells and CHO cell mutants deficient in GAG synthesis at an MOI of 10 for 1 h at 37xc2x0 C. Cells were harvested 44 h post infection and assayed for xcex2-galactosidase activity. Data represent the mean and standard deviation of an experiment performed in triplicate.
FIG. 8 demonstrates the correlation between relative HS expression levels and AAV-2 binding. Relative percent of cell surface heparan sulfate (HS) (left-hand bar) compared to AAV-2 binding (right-hand bar) to a panel of in vitro cell lines (CHO cells=100%). Cell surface heparan sulfate was determined by FACS analysis using anti-heparan sulfate HepSS-1 monoclonal antibody. Relative HS expression was determined by taking the fold difference between the median fluorescence obtained with a control antibody and HepSS-1. Binding studies were performed with 3H-labeled AAV-2 virions at 4xc2x0 C.
FIGS. 9A and 9B illustrate that screening of cell samples for the presence of the AAV receptor is predictive of the ability of cells to bind AAV. FIG. 9A, illustrates human bone marrow CD34+ cells positive for both AAV virus binding (top graph) and heparan sulfate (bottom graph). Fluorescently-labeled AAV or anti-heparan sulfate antibody (FITC) were incubated with cells for one hour at 4xc2x0 C. Cells were washed three times and fixed in a 1% paraformaldehyde solution prior to FACS scan. The results are overlaid onto control samples with unlabelled virus or non-specific FITC-conjugated antibody. As is seen in FIG. 9A, cells that are positive for the AAV receptor cell surface heparan sulfate exhibit a shift in relative fluorescent value to the right (bottom graph), as compared to non-specific FITC conjugated antibody. Similarly, virus bound to the cell surface exhibit a spectroscopic shift to the right when compared to unlabelled virus. FIG. 9B illustrates a FACS analysis screen for cells that are negative for both cell surface heparan sulfate (i.e., antibody specific for heparan sulfate does not bind to the cell) and for AAV virus binding. When the control data are overlaid onto the experimental data, no fluorescent shift is observed.
FIG. 10 illustrates the elution profile of AAV purified using affinity chromatography with heparin bound to a solid support. Results are shown as virus titer expressed in BFU/ml.
FIGS. 11A and 11B illustrate the effect of EDTA on Ad and AAV infection. HeLa cells were infected with recombinant virus (rAd-LacZ [FIG. 11A], rAAV-LacZ [FIG. 11B]) at an MOI of 2 either in the presence or absence of divalent cation chelator EDTA (20 mM) as described in methods. 24 h post Ad infection, and 36 h post AAV infection, cells were fixed and stained for xcex2-galactosidase activity (upper panel). Transduction has been quantitated in the lower panel as the percentage of HeLa cells transduced in the presence or absence of EDTA.
FIG. 12 illustrates viral overlay and western blot analysis of plasma membrane proteins. Panel A: Purified HeLa cell plasma membrane proteins were separated by 5-20% gradient SDS-PAGE under reducing conditions. After blotting to nitrocellulose, proteins were probed with either no virus (lane 1), purified AAV-2 virions (lane 2), or B5-IVF2 mAb for detection of the xcex25 subunit of xcex1vxcex25 integrin (lane 3). Blots were then incubated with A20 mAb, which interacts with AAV-2 virions (lanes 1 and 2) and secondary goat anti-mouse IgG conjugated to HRP (lanes 1, 2, and 3) for detection by chemiluminescence and autoradiography. Panel B: Viral overlay analysis of two different membrane preparations. Lanes 1 and 2 represent an AAV-2 overlay. Arrows point to the 150 kDa (lane 1) and 100 kDa (lane 2) proteins that interact with AAV. The corresponding control overlays performed without virus are shown in lanes 3 and 4.
FIG. 13 illustrates a virus overlay of immunoprecipitated xcex25 subunit of xcex1vxcex25 integrin (Panels A and B). Purified plasma membrane proteins (lanes 1, Panels A and B), immunoprecipitated xcex25 subunit of xcex1vxcex25 integrin (lanes 2, Panels A and B), and control immunoprecipitations [isotype matched IgG1 Ab (Panels A and B, lanes 4), or rabbit anti-mouse Ab (Panels A and B, lanes 3)] were separated by 7.5% SDS-PAGE under reducing conditions. Proteins were blotted to nitrocellulose and probed with (Panel A) or without (Panel B) purified AAV-2 virions as described in FIG. 12.
FIGS. 14A, 14B, and 14C illustrate xcex1vxcex25 expression and vector transduction of CS-1 and CS1/xcex25 cells. The data in FIG. 14A demonstrates FAGS analysis of avb5 expression on CS-1 and CS1/xcex25 cell lines. xcex1vxcex25 integrin was identified with PIF6 mAb using mouse IgG1 (MOPC 21) as control isotype matched Ab. Transduction of CS-1 and CS1/xcex25 cell lines with rAAV (FIG. 14B), or rAd (FIG. 14C). Gene transduction was determined by a chemiluminescence assay 24 h (rAd) or 48 h (rAAV) post infection. Data represent the mean and standard deviation of experiments performed in triplicate (FIGS. 14B and 14C). Separate experiments yielded the same results.
FIG. 15 demonstrates binding of AAV-2 to CS-1 and CS1/xcex25 cells. Direct binding assays were performed with 3H-wt AAV. Briefly, 4xc3x971011 labeled virus particles were incubated with 3xc3x97105 cells at 4xc2x0 C. for 90 min. After extensive washing, cells were solubilized in 0.3 N NaOH and neutralized with glacial acetic acid prior to measuring cell associated radioactivity in a scintillation counter. Non-specific binding was determined in the presence of a 50 fold excess of unlabeled wt AAV (right-hand column). Data are the mean and standard deviation of two experiments performed in duplicate.
FIG. 16 illustrates cell surface expression of HS on CS-1 and CS1/xcex25 cells. Flow cytometric analysis of CS-1 (Panel A) and CS1/xcex25 (Panel B) cells was performed with monoclonal antibody HepSS-1 to detect cell surface expression of heparan sulfate. Results are overlaid onto fluorescence intensity histograms obtained with an isotype matched control antibody. CS-1 and CS1/xcex25 cells express similar levels of HS.
FIG. 17 illustrates Internalization of Cy3-AAV2 by CS-1 and CS1/xcex25 cells. Cells were incubated with Cy3-AAV2 for 1 h at 4xc2x0 C., washed extensively, and then transferred to 37xc2x0 C. to allow virus internalization. At the indicated times, cells were fixed and prepared for microscopy. Images were obtained by confocal microscopy from cross sections representative of the cells center. The CS1/xcex25 cell line shows a clear increase in the rate of internalization of the fluorescent virus relative to the CS1 cell line. Independent experiments yielded similar results.